Modulation of hepatitis B virus cccDNA transcription

ABSTRACT

The present invention provides small molecule inhibitors of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, which are useful as therapeutics in the management of chronic HBV. The compounds of the invention achieve epigenetic modification of the cccDNA, histone modification and histone deacetylase activity inhibition, thus modulating HBV cccDNA. The present invention further provides methods for modulating HBV cccDNA, for treating or preventing HBV in a subject, and for modulating cccDNA transcription of hepatitis B in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 14/435,675, filed Apr. 14, 2015, now issuedas U.S. Pat. No. 9,623,071, which is the U.S. National Phase Applicationfiled under 35 U.S.C. § 371 claiming priority to PCT InternationalApplication No. PCT/US2013/043691, filed May 31, 2013, which claimspriority to U.S. Provisional Application No. 61/654,374, filed Jun. 1,2012, all of which applications are hereby incorporated herein byreference in their entireties.

TECHNICAL FIELD

The present disclosure pertains to the use of pharmacological agents,preferably with histone deacetylase activity, for modulating covalentlyclosed circular DNA of hepatitis B virus, and for preventing or treatinghepatitis B.

BACKGROUND

There are now seven medications approved by the United States Food &Drug Administration (FDA) for the management of chronic hepatitis B,which fall into one of two categories: the interferons (IFNs) and thepolymerase inhibitors (Lok, A. S., and B. J. McMahon. 2007. ChronicHepatitis B. Hepatology 45:507-539). These are recommended for use inapproximately 50% or less of the infected population of more than 350million. Although this is the highest risk population, those who falloutside the treatment guidelines may also benefit from intervention,since they are also at significantly elevated risk of liver diseases.The IFNs are limited by significant side effects. The pol inhibitorstarget the same viral life cycle step and thus combination therapy, thebulwark of HIV and curative HCV therapy, is of limited value. Theyrequire lifelong use, and are subject to eventual use limitingtoxicities, as seen with HIV long term medication use, and the emergenceof drug resistant mutants. Thus, alternatives and complements to thecurrent portfolio of medications are needed.

There is a growing belief that a “cure”, or at least sustained off-drugcontrol of HBV, will require, or at least benefit from, drugs thatcontrol the viral nuclear genome, the covalently closed circular DNA(cccDNA). The 2006 NIDDK Liver Action Plan, reinforced by the 2010Institute of Medicine report, all call for cccDNA inhibition as apriority for HBV drug development.

However, screening for HBV cccDNA inhibitors has been difficult, becauseof technical reasons: HBV cccDNA is made in amounts to low to beconveniently detected, and most viral gene products in conventionallytransfected cells in culture are derived from transgenes of the viralgenome, not cccDNA. The present inventors have created cell lines inwhich HBV gene products such as the HBeAg are produced only from cccDNA,but not from integrated viral transgene and in amounts to be robustlydetected, making screening realistic (Cai, D., et al., 2012.Identification of the Disubstituted Sulfonamide Compounds as SpecificInhibitors of Hepatitis B Virus Covalently Closed Circular DNAFormation. Antimicrobial Agents and Chemotherapy: In Press; Zhou, T, etal., 2006. Hepatitis B virus e antigen production is dependent uponcovalently closed circular (ccc) DNA in HepAD38 cell cultures and mayserve as a cccDNA surrogate in antiviral screening assays. AntiviralResearch 72:116-124).

Given such challenges, it is unsurprising that there are no HBVtherapeutics in use that target HBV cccDNA and, there have been few, ifany, programs to screen and develop cccDNA inhibitors. This is largelydue to technical difficulties (see Block, T M, et al. 2003. Molecularviral oncology of hepatocellular carcinoma. Oncogene 22:5093-5107;Locarnini, S. 2005. Therapies for hepatitis B: where to from here?Gastroenterology 128: 789-792; Lok, A. S. 2011. Does antiviral therapyfor hepatitis B and C prevent hepatocellular carcinoma? J GastroenterolHepatol 26:221-227). In addition, the role of host functions inregulating HBV cccDNA transcription and stability is poorly understoodfurther frustrating development of therapeutics. Thus, any work in thisarea would be innovative, and would address the outstanding andlong-felt need for drugs that control the viral nuclear genome ofhepatitis B and otherwise provide treatment for HBV infection.

SUMMARY

Provided are methods of modulating cccDNA transcription of hepatitis Bin a subject comprising administering to the subject an agent thatprovides epigenetic modification of the cccDNA, a histone modifyingagent, or an inhibitor of histone deacetylase activity. For example, theepigenetic modifying agent, histone modifying agent, or inhibitor ofhistone deacetylase activity may be pharmacological, such as a smallmolecule.

Also provided are methods of treating hepatitis B in a subjectcomprising administering to the subject an inhibitor of histonedeacetylase activity.

The present disclosure also pertains to method of modulating hepatitis Bvirus covalently closed circular DNA comprising contacting a hepatitis Bvirus with an inhibitor of histone deacetylase activity.

Also disclosed are compounds according to formula II:

wherein R₁-R₈ are defined as provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides data demonstrating that HBV cccDNA is efficiently formedand transcriptionally active in dstet5 cells.

FIG. 2 relates to experiments demonstrating that cccDNA can be inhibitedby IFN-a.

FIG. 3 pertains to the present finding that Apicidin and TSA represscccDNA transcription.

FIG. 4 relates to the discovery that HDAC inhibitors dose-dependentlystimulate DHBV pgRNA synthesis from transgene integrated in a hostcellular chromosome.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingfigures and examples, which form a part this disclosure. It is to beunderstood that this invention is not limited to the specific products,methods, conditions or parameters described and/or shown herein, andthat the terminology used herein is for the purpose of describingparticular embodiments by way of example only and is not intended to belimiting of the claimed invention.

The disclosures of each patent, patent application, and publicationcited or described in this document are hereby incorporated herein byreference, in their entirety.

As employed above and throughout the disclosure, the following terms andabbreviations, unless otherwise indicated, shall be understood to havethe following meanings.

In the present disclosure the singular forms “a,” “an,” and “the”include the plural reference, and reference to a particular numericalvalue includes at least that particular value, unless the contextclearly indicates otherwise. Thus, for example, a reference to “acompound” is a reference to one or more of such compounds andequivalents thereof known to those skilled in the art, and so forth.Furthermore, when indicating that a certain chemical moiety “may be” X,Y, or Z, it is not intended by such usage to exclude in all instancesother choices for the moiety; for example, a statement to the effectthat R₁ “may be alkyl, aryl, or amino” does not necessarily excludeother choices for R₁, such as halo, aralkyl, and the like.

When values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment. As used herein, “about X” (where X is a numerical value)preferably refers to ±10% of the recited value, inclusive. For example,the phrase “about 8” refers to a value of 7.2 to 8.8, inclusive; asanother example, the phrase “about 8%” refers to a value of 7.2% to8.8%, inclusive. Where present, all ranges are inclusive and combinable.For example, when a range of “1 to 5” is recited, the recited rangeshould be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2& 4-5”, “1-3 & 5”, and the like. In addition, when a list ofalternatives is positively provided, such listing can be interpreted tomean that any of the alternatives may be excluded, e.g., by a negativelimitation in the claims. For example, when a range of “1 to 5” isrecited, the recited range may be construed as including situationswhereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, arecitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, orsimply “wherein 2 is not included.” In another example, when a listingof possible substituents including “hydrogen, alkyl, and aryl” or““hydrogen, alkyl, or aryl” is provided, the recited listing may beconstrued as including situations whereby any of “hydrogen, alkyl, andaryl” or “hydrogen, alkyl, or aryl” is negatively excluded; thus, arecitation of “hydrogen, alkyl, and aryl” or “hydrogen, alkyl, and aryl”may be construed as “hydrogen and/or aryl, but not alkyl”, or simply“wherein the substituent is not alkyl”.

As used herein, the terms “component,” “composition of compounds,”“compound,” “drug,” “pharmacologically active agent,” “active agent,”“therapeutic,” “therapy,” “treatment,” or “medicament” are usedinterchangeably herein to refer to a compound or compounds orcomposition of matter which, when administered to a subject (human oranimal) induces a desired pharmacological and/or physiologic effect bylocal and/or systemic action.

The abbreviations in the specification correspond to units of measure,techniques, properties, or compounds as follows: “min” means minute(s),“g” means gram(s), “mg” means milligram(s), “μg” means microgram(s),“eq” means equivalent(s), “h” means hour(s), “μL” means microliter(s),“mL” means milliliter(s), “mM” means millimolar, “M” means molar, “mmol”or “mmole” means millimole(s), “cm” means centimeters, “SEM” meansstandard error of the mean, and “IU” means International Units. “IC₅₀value” or “IC₅₀” means dose of the compound which results in 50%alleviation or inhibition of the observed condition or effect.

“Apicidin” is a compound derived from a Fusarium species fungalmetabolite. It has the structurecyclo(N—O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl).

“Natural analogs of Apicidin” refers to analogs of Apicidin that areproduced in fermentations of Fusarium pallidoroseum species ATCC74322and ATCC47289 (Apicidins A, B, C, D1, D2, D3, which are described in JOC67, 815 (2002) and Tet Lett, 37, 8077 (1996), and in WO 1996/9603428.

As used herein, “alkyl” refers to an optionally substituted, saturatedstraight, or branched, hydrocarbon radical having from about 1 to about20 carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein). Where appropriate, “alkyl”can mean “alkylene”; for example, if X is and R₁ is said to be “alkyl”,then “alkyl” may correctly be interpreted to mean “alkylene”.

“Amino” refers to —NH₂ and may include one or more substituents thatreplace hydrogen. “Amino” is used interchangeably with amine and is alsointended to include any pharmaceutically acceptable amine salts. Forexample, amino may refer to —NH⁺(X)(Y)Cl⁻, wherein X and Y arepreferably and independently hydrogen or alkyl, wherein alkyl mayinclude one or more halo substitutions.

As used herein, “aryl”, “arene”, and “aromatic” each refer to anoptionally substituted, saturated or unsaturated, monocyclic,polycyclic, or other homo-, carbo- or heterocyclic aromatic ring systemhaving from about 3 to about 50 ring members (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein),with from about 5 to about 10 ring atom members being preferred. Suchmoieties encompass (include) “heteroaryl” and “heteroarene” as definedinfra. Where appropriate, “aryl” can mean “arene”; for example, if X is—R₁R₂, and R₁ is said to be “aryl”, then “aryl” may correctly beinterpreted to mean “arene”.

As used herein, “alkenyl” refers to an alkyl radical having from about 2to about 20 carbon atoms and one or more double bonds (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), wherein alkyl is as previously defined. In someembodiments, it is preferred that the alkenyl groups have from about 2to about 6 carbon atoms. Alkenyl groups may be optionally substituted.

As used herein, “aralkyl” refers to alkyl radicals bearing one or morearyl substituents and having from about 4 to about 50 carbon atoms (andall combinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), wherein aryl and alkyl are as previously defined.In some preferred embodiments, the alkyl moieties of the aralkyl groupshave from about 1 to about 4 carbon atoms. In other preferredembodiments, the alkyl moieties have from about 1 to about 3 carbonatoms. Aralkyl groups may be optionally substituted.

“Alkylamino” signifies alkyl-(NH)—, wherein alkyl is as previouslydescribed and NH is defined in accordance with the provided definitionof amino. “Arylamino” represents aryl-(NH)—, wherein aryl is as definedherein and NH is defined in accordance with the provided definition ofamino. Likewise, “aralkylamino” is used to denote aralkyl-(NH)—, whereinaralkyl is as previously defined and NH is defined in accordance withthe provided definition of amino. “Alkylamido” refers toalkyl-CH(═O)NH—, wherein alkyl is as previously described. “Alkoxy” asused herein refers to the group R—O— where R is an alkyl group, andalkyl is as previously described. “Aralkoxy” stands for R—O—, wherein Ris an aralkyl group as previously defined. “Alkylsulfonyl” meansalkyl-SO₂—, wherein alkyl is as previously defined. “Aminooxy” as usedherein refers to the group amino-(O)—, wherein amino is defined asabove. “Aralkylaminooxy” as used herein is used to denotearyl-akyl-aminooxy-, wherein aryl, alkyl, and aminooxy are respectivelydefined as provided previously.

As used herein, “alkylene” refers to an optionally branched orsubstituted bivalent alkyl radical having the general formula—(CH₂)_(n)—, where n is 1 to 10. Non-limiting examples includemethylene, trimethylene, pentamethylene, and hexamethylene.

“Alkyleneamino” refers to —(CH₂)_(n)—NH—, where n is 1 to 10 and whereinthe bivalent alkyl radical may be optionally branched or substituted,and the amino group may include one or more substituents that replacehydrogen.

As used herein, “heteroaryl” or “heteroarene” refers to an aryl radicalwherein in at least one of the rings, one or more of the carbon atomring members is independently replaced by a heteroatom group selectedfrom the group consisting of S, O, N, and NH, wherein aryl is aspreviously defined. Heteroaryl/heteroarene groups having a total of fromabout 3 to about 14 carbon atom ring members and heteroatom ring membersare preferred. Likewise, a “heterocyclic ring” is an aryl radicalwherein one or more of the carbon atom ring members may be (but are notnecessarily) independently replaced by a heteroatom group selected fromthe group consisting of S, O, N, and NH. Heterocyclic rings having atotal from about 3 to 14 ring members and heteroatom ring members arepreferred, but not necessarily present; for example, “heterocyclohexyl”may be a six-membered aryl radical with or without a heteroatom group.

“Halo” and “halogen” each refers to a fluoro, chloro, bromo, or iodomoiety, with fluoro, chloro, or bromo being preferred.

“Haloalkyl” signifies halo-alkyl- wherein alkyl and halo, respectively,are as previously described.

The phrase reading “[moiety] is absent” may mean that the substituentsto which the moiety is attached are directly attached to each other.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen. Exemplary substituents include, forexample, halo (e.g., F, Cl, Br, I), alkyl, cycloalkyl, alkylcycloalkyl,cycloalkylalkyl, alkenyl, alkynyl, aralkyl, aryl, heteroaryl,heteroaralkyl, spiroalkyl, heterocycloalkyl, hydroxyl (—OH), nitro(—NO₂), cyano (—CN), amino (—NH₂), —N-substituted amino (—NHR″),—N,N-disubstituted amino (—N(R″)R″), oxo (═O), carboxy (—COOH),—O—C(═O)R″, —C(═O)R″, —OR″, —C(═O)OR″, -(alkylene)-C(═O)—OR″,—NHC(═O)R″, aminocarbonyl (—C(═O)NH₂), —N-substituted aminocarbonyl(—C(═O)NHR″), —N,N-disubstituted aminocarbonyl (—C(═O)N(R″)R″), thiol,thiolato (—SR″), sulfonic acid (—SO₃H), phosphonic acid (—PO₃H),—P(═O)(OR″)OR″, —S(═O)R″, —S(═O)₂R″, —S(═O)₂NH₂, —S(═O)₂ NHR″,—S(═O)₂NR″R″, —NHS(═O)₂R″, —NR″S(═O)₂R″, —CF₃, —CF₂CF₃, —NHC(═O)NHR″,—NHC(═O)NR″R″, —NR″C(═O)NHR″, —NR″C(═O)NR″R″, —NR″C(═O)R″ and the like.In relation to the aforementioned substituents, each moiety R″ can be,independently, any of H, alkyl, cycloalkyl, alkenyl, aryl, aralkyl,heteroaryl, or heterocycloalkyl, for example.

As used herein, the terms “treatment” or “therapy” (as well as differentword forms thereof) includes preventative (e.g., prophylactic), curativeor palliative treatment.

As employed above and throughout the disclosure the term “effectiveamount” refers to an amount effective, at dosages, and for periods oftime necessary, to achieve the desired result with respect to thetreatment of the relevant disorder, condition, or side effect. It willbe appreciated that the effective amount of components of the presentinvention will vary from patient to patient not only with the particularcompound, component or composition selected, the route ofadministration, and the ability of the components to elicit a desiredresponse in the individual, but also with factors such as the diseasestate or severity of the condition to be alleviated, hormone levels,age, sex, weight of the individual, the state of being of the patient,and the severity of the pathological condition being treated, concurrentmedication or special diets then being followed by the particularpatient, and other factors which those skilled in the art willrecognize, with the appropriate dosage ultimately being at thediscretion of the attendant physician. Dosage regimens may be adjustedto provide the improved therapeutic response. An effective amount isalso one in which any toxic or detrimental effects of the components areoutweighed by the therapeutically beneficial effects. As an example, thecompounds useful in the methods of the present invention areadministered at a dosage and for a time such that the level ofactivation and adhesion activity of platelets is reduced as compared tothe level of activity before the start of treatment.

“Pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem complications commensurate with a reasonablebenefit/risk ratio.

Within the present invention, the disclosed compounds may be prepared inthe form of pharmaceutically acceptable salts. “Pharmaceuticallyacceptable salts” refer to derivatives of the disclosed compoundswherein the parent compound is modified by making acid or base saltsthereof. Examples of pharmaceutically acceptable salts include, but arenot limited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts include theconventional non-toxic salts or the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. For example, such conventional non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Thesephysiologically acceptable salts are prepared by methods known in theart, e.g., by dissolving the free amine bases with an excess of the acidin aqueous alcohol, or neutralizing a free carboxylic acid with analkali metal base such as a hydroxide, or with an amine.

Compounds described herein throughout, can be used or prepared inalternate forms. For example, many amino-containing compounds can beused or prepared as an acid addition salt. Often such salts improveisolation and handling properties of the compound. For example,depending on the reagents, reaction conditions and the like, compoundsas described herein can be used or prepared, for example, as theirhydrochloride or tosylate salts. Isomorphic crystalline forms, allchiral and racemic forms, N-oxide, hydrates, solvates, and acid salthydrates, are also contemplated to be within the scope of the presentinvention.

Certain acidic or basic compounds of the present invention may exist aszwitterions. All forms of the compounds, including free acid, free baseand zwitterions, are contemplated to be within the scope of the presentinvention. It is well known in art that compounds containing both aminoand carboxy groups often exist in equilibrium with their zwitterionicforms. Thus, any of the compounds described herein throughout thatcontain, for example, both amino and carboxy groups, also includereference to their corresponding zwitterions.

“Hydrate” refers to a compound of the present invention which isassociated with water in the molecular form, i.e., in which the H—OHbond is not split, and may be represented, for example, by the formulaR.H₂O, where R is a compound of the invention. A given compound may formmore than one hydrate including, for example, monohydrates (R.H₂O) orpolyhydrates (R.nH₂O wherein n is an integer>1) including, for example,dihydrates (R.2H₂O), trihydrates (R.3H₂O), and the like, orhemihydrates, such as, for example, R.n_(/2)H₂O, R.n_(/3)H₂O,R.n_(/4)H₂O and the like wherein n is an integer.

“Solvate” refers to a compound of the present invention which isassociated with solvent in the molecular form, i.e., in which thesolvent is coordinatively bound, and may be represented, for example, bythe formula R-(solvent), where R is a compound of the invention. A givencompound may form more than one solvate including, for example,monosolvates (R.(solvent)) or polysolvates (R.n(solvent)) wherein n isan integer>1) including, for example, disolvates (R.2(solvent)),trisolvates (R.3(solvent)), and the like, or hemisolvates, such as, forexample, R.n_(/2)(solvent), R.n_(/3)(solvent), R.n_(/4)(solvent) and thelike wherein n is an integer. Solvents herein include mixed solvents,for example, methanol/water, and as such, the solvates may incorporateone or more solvents within the solvate.

“Acid hydrate” refers to a complex that may be formed throughassociation of a compound having one or more base moieties with at leastone compound having one or more acid moieties or through association ofa compound having one or more acid moieties with at least one compoundhaving one or more base moieties, said complex being further associatedwith water molecules so as to form a hydrate, wherein said hydrate is aspreviously defined and R represents the complex herein described above.

The term “stereoisomers” refers to compounds that have identicalchemical constitution, but differ as regards the arrangement of theatoms or groups in space.

“Racemic” means having the capacity for resolution into forms of opposedoptical activity.

As used herein, the term “partial stereoisomer” refers to stereoisomershaving two or more chiral centers wherein at least one of the chiralcenters has defined stereochemistry (i.e., R or S) and at least one hasundefined stereochemistry (i.e., R or S). When the term “partialstereoisomers thereof” is used herein, it refers to any compound withinthe described genus whose configuration at chiral centers with definedstereochemistry centers is maintained and the configuration of eachundefined chiral center is independently selected from R or S. Forexample, if a stereoisomer has three chiral centers and thestereochemical configuration of the first center is defined as having“S” stereochemistry, the term “or partial stereoisomer thereof” refersto stereoisomers having SRR, SRS, SSR, or SSS configurations at thethree chiral centers, and mixtures thereof.

An “isotopically substituted analogue” is a compound of the presentdisclosure in which one or more atoms have been replaced with an isotopeof that atom. For example, hydrogen (protium) may be substituted withdeuterium or tritium. Other atoms that may be replaced with an isotopethereof in order to form an isotopically substituted analogue thereofinclude, for example, carbon (replaced with C¹³), nitrogen (replacedwith N¹⁵), iodine (replaced with I¹³¹), fluorine (replaced with F¹⁸), orsulfur (replaced with S³¹). Any available isotope may be used to form anisotopically substituted analogue thereof, and those of ordinary skillin the art will recognize available techniques for forming suchanalogues from a given compound.

“Prodrug” refers to compounds which are themselves inactive or minimallyactive for the activity desired, but through biotransformation can beconverted into biologically active metabolites. For example, a prodrugof the present invention would include, inter alia, any compound whichis convertible in vivo by metabolic means to a compound claimed ordescribed in the present disclosure.

“N-oxide” refers to compounds wherein the basic nitrogen atom of eithera heteroaromatic ring or tertiary amine is oxidized to give a quaternarynitrogen bearing a positive formal charge and an attached oxygen atombearing a negative formal charge.

When any variable occurs more than one time in any constituent or in anyformula, its definition in each occurrence is independent of itsdefinition at every other occurrence. Combinations of substituentsand/or variables are permissible only if such combinations result instable compounds.

The term “administering” means either directly administering a compoundor composition of the present invention, or administering a prodrug,derivative or analog which will form an equivalent amount of the activecompound or substance within the body.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit maycontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention may be dictated by (a) the unique characteristicsof the active compound(s) and the particular therapeutic effect(s) to beachieved, and (b) the limitations inherent in the art of compoundingsuch active compound(s).

“Subject” or “patient” refers to an embryonic, immature, or adultanimal, including the human species, that is treatable with thecompositions, and/or methods of the present invention.

It has presently been discovered that hepatitis B virus covalentlyclosed circular DNA (cccDNA), existing and being expressed as an“episome” in the nucleus of an infected cell, is regulated differentlythan HBV DNA integrated in to the host chromosome, and that RNAexpression from the HBV cccDNA can be pharmacologically suppressed,selectively, as compared to other genes (as described more fullyherein). Indeed, the present inventors have identified numerouscompounds that repress DHBV cccDNA transcription in a reproducible androbust manner, and that occurs at low concentrations and underconditions of no apparent toxicity. These results represent the firsttime that selective pharmacological suppression has been achieved, bydesign, with small molecules. The result that gene expression from HBVcccDNA is regulated differently that the same or similar DNA integratedin to the host chromosomes is surprising and a highly usefulobservation, in that it enables therapies that selectively represscccDNA DNA (for example, as compared with integrated HBV DNA) withoutsuppressing or otherwise affecting host chromosomal DNA. The presentfinding that HBV cccDNA can be suppressed pharmacologically washeretofore unknown, and offers the proof of useful concept of the priorstatement, and demonstrates that such pharmacological suppression ispossible.

Accordingly, the present disclosure provides, inter alia, methods ofmodulating cccDNA transcription of hepatitis B in a subject comprisingadministering to the subject an agent that provides epigeneticmodification of the cccDNA, a histone modifying agent, or an inhibitorof histone deacetylase activity. For example, the epigenetic modifyingagent, histone modifying agent, or inhibitor of histone deacetylaseactivity may be pharmacological, such as a small molecule. Theepigenetic modifying agent, histone modifying agent, or inhibitor ofhistone deacetylase activity may be selective for the inhibition ofcccDNA, as compared with integrated HBV DNA, i.e., does not inhibitintegrated HBV DNA, and/or as compared with cellular host DNA, i.e.,does not inhibit cellular host DNA. The inhibitor of histone deacetylaseactivity may be an inhibitor of multiple classes of histone deacetylase,or may be selective for a particular class of histone deacetylase. Forexample, the inhibitor may be an inhibitor of class I histonedeacetylase activity, class II histone deacetylase activity, or both.Preferably, the inhibitor of histone deacetylase activity is aninhibitor of class I histone deacetylase activity. Numerous inhibitorsof histone deacetylase activity are known, and any such HDAC inhibitormay be used pursuant to the present methods.

The present methods of modulating cccDNA transcription of hepatitis Bmay also include—in addition to the administration to the subject anagent that provides epigenetic modification of the cccDNA, a histonemodifying agent, or an inhibitor of histone deacetylaseactivity—administering to the subject a therapeutically effective amountof a further agent that modulates hepatitis B virus. The further agentmay be administered simultaneously with, or simply as a part of the samegeneral therapy regimen as the agent that provides epigeneticmodification of the cccDNA, histone modifying agent, or inhibitor ofhistone deacetylase activity. The further agent may be any substancethat is presently used for modulation of HBV, of which numerous typesare known among those skill in the art. For example, existing drugs forthe modulation of HBV include interferons (e.g., interferon alpha,pegylated interferon), nucleoside analogues (e.g., lamivudine, adefovirdipivoxil, entecavir, telbivudine, tenofovir, clevudine, amdoxovir),non-nucleoside antivirals (e.g., BAM 205, ANA380, myrcludex B, HAPCompound Bay 41-4109, REP 9AC, nitazoxanide, dd-RNAi compound, ARC-520,NVR-1221), non-interferon immune enhancers (e.g., thymosin alpha-1,interleukin-7, DV-601, HBV core antigen vaccine, GS-9620, GI13000), andpost-exposure and/or post-liver transplant treatment drugs (e.g.,hyperHEP S/D, Nabi-GB, Hepa Gam B).

In particular, the further agent may be any other Direct ActingAntiviral anti hepatitis B agent (such as the polymerase inhibitorsBarraclude, Tenofovir, lamivudine, telbivudine, and adefovir) and/or anyother directing acting antiviral agents that work at a step in the viruslife cycle other than suppression of cccDNA transcription, such ascapsid inhibitors, secretion inhibitors, or entry inhibitors. Thefurther agent may also be any other non-direct acting antiviral agent,such as an interferon or other immunomodulatory agent.

In accordance with the present methods of modulating cccDNAtranscription of hepatitis B, the inhibitor of histone deacetylaseactivity may be, for example, Trichostatin A, suberoyl bis hydroxamicacid, 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl] benzamide,Apicidin, an Apicidin analog (for example, a natural analog of Apicidinor an analog that is synthesized de novo), or a compound according toformula (I)

wherein

R₁ is —(CH₂)_(n)— or —C(═O)—;

R₂ is —C(═O)—, 3,5-triazolyl, or —C(Z)N(R₄)—;

R₄ is hydrogen, alkyl, aryl, aralkyl, dialkylaminoalkyl, orcarboxyalkyl;

R₃ is —CH(R₅)—, or R₂ is nitrogen and R₃ is —CH— and R₂ and R₃ togetherform piperidinyl;

R₅ is hydrogen, —CH₃, or an alpha amino acid R group;

R₆ is —(CH₂)_(m)C(X)Y, —(CH₂)₂CH₃, or—(CH₂)_(q)-phenyl-(CH₂)_(m)C(═O)NHOH;

X is ═O, H₂, ═N—NH₂, or ═N—NH—C(═O)NH₂;

Y is NHOH or —CH₂CH₃;

Z is H₂ or O;

R₇ is hydrogen or alkoxy;

R₈ is alkyl or carboxyalkyl;

n is 0-2;

m is 0-6; and,

q is 0-3;

or a stereoisomer or pharmaceutically acceptable salt thereof.

As used herein, the phrase “alpha amino acid R group” refers to a sidechain group from a natural or unnatural amino acid.

In certain embodiments, the inhibitor of histone deacetylase activity isApicidin,

wherein

-   -   R₁ is —(CH₂)—,    -   and,    -   R₂ is —C(Z)N(R₄)—        or a stereoisomer or pharmaceutically acceptable salt thereof.

In other embodiments, the inhibitor of histone deacetylase activity is

or a stereoisomer or pharmaceutically acceptable salt thereof.

The present disclosure also pertains to methods of treating hepatitis Bin a subject comprising administering to the subject an agent thatprovides epigenetic modification of the cccDNA, a histone modifyingagent, or an inhibitor of histone deacetylase activity. For example, theepigenetic modifying agent, histone modifying agent, or inhibitor ofhistone deacetylase activity may be pharmacological, such as a smallmolecule. The epigenetic modifying agent, histone modifying agent, orinhibitor of histone deacetylase activity may be selective for theinhibition of cccDNA, as compared with integrated HBV DNA, i.e., doesnot inhibit integrated HBV DNA, and/or as compared with cellular hostDNA, i.e., does not inhibit cellular host DNA. The inhibitor of histonedeacetylase activity may be an inhibitor of multiple classes of histonedeacetylase, or may be selective for a particular class of histonedeacetylase. For example, the inhibitor may be an inhibitor of class Ihistone deacetylase activity, class II histone deacetylase activity, orboth. Preferably, the inhibitor of histone deacetylase activity is aninhibitor of class I histone deacetylase activity. Numerous inhibitorsof histone deacetylase activity are known, and any such HDAC inhibitormay be used pursuant to the present methods.

In accordance with the present methods of treating hepatitis B in asubject, the inhibitor of histone deacetylase activity may be, forexample, Trichostatin A, suberoyl bis hydroxamic acid,4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide, Apicidin,an Apicidin analog (for example, a natural analog of Apicidin or ananalog that is synthesized de novo), or a compound according to formula(I)

wherein

R₁ is —(CH₂)_(n)— or —C(═O)—;

R₂ is —C(═O)—, 3,5-triazolyl, or —C(Z)N(R₄)—;

R₄ is hydrogen, alkyl, aryl, aralkyl, dialkylaminoalkyl, orcarboxyalkyl;

R₃ is —CH(R₅)—, or R₂ is nitrogen and R₃ is —CH— and R₂ and R₃ togetherform piperidinyl;

R₅ is hydrogen, —CH₃, or an alpha amino acid R group;

R₆ is —(CH₂)_(m)C(X)Y, —(CH₂)₂CH₃, or—(CH₂)_(q)-phenyl-(CH₂)_(m)C(═O)NHOH;

X is ═O, H₂, ═N—NH₂, or ═N—NH—C(═O)NH₂;

Y is NHOH or —CH₂CH₃;

Z is H₂ or O;

R₇ is hydrogen or alkoxy;

R₈ is alkyl or carboxyalkyl;

n is 0-2;

m is 0-6; and,

q is 0-3;

or a stereoisomer or pharmaceutically acceptable salt thereof.

In certain embodiments, the inhibitor of histone deacetylase activity isApicidin,

wherein

-   -   R₁ is —(CH₂)—,    -   and,    -   R₂ is —C(Z)N(R₄)—        or a stereoisomer or pharmaceutically acceptable salt thereof.

In other embodiments, the inhibitor of histone deacetylase activity is

or a stereoisomer or pharmaceutically acceptable salt thereof.

The present methods of treating hepatitis B in a subject may alsoinclude—in addition to the administration to the subject an agent thatprovides epigenetic modification of the cccDNA, a histone modifyingagent, or an inhibitor of histone deacetylase activity—administering tothe subject a therapeutically effective amount of a further agent thatmodulates hepatitis B virus. The further agent may be administeredsimultaneously with, or simply as a part of the same general therapyregimen as the agent that provides epigenetic modification of thecccDNA, histone modifying agent, or inhibitor of histone deacetylaseactivity. The further agent may be any substance that is presently usedfor modulation of HBV, of which numerous types are known among thoseskill in the art. For example, existing drugs for the modulation of HBVinclude interferons (e.g., interferon alpha, pegylated interferon),nucleoside analogues (e.g., lamivudine, adefovir dipivoxil, entecavir,telbivudine, tenofovir, clevudine, amdoxovir), non-nucleoside antivirals(e.g., BAM 205, ANA380, myrcludex B, HAP Compound Bay 41-4109, REP 9AC,nitazoxanide, dd-RNAi compound, ARC-520, NVR-1221), non-interferonimmune enhancers (e.g., thymosin alpha-1, interleukin-7, DV-601, HBVcore antigen vaccine, GS-9620, GI13000), and post-exposure and/orpost-liver transplant treatment drugs (e.g., hyperHEP S/D, Nabi-GB, HepaGam B).

In particular, the further agent may be any other Direct ActingAntiviral anti hepatitis B agent (such as the polymerase inhibitorsBarraclude, Tenofovir, lamivudine, telbivudine, and adefovir) and/or anyother directing acting antiviral agents that work at a step in the viruslife cycle other than suppression of cccDNA transcription, such ascapsid inhibitors, secretion inhibitors, or entry inhibitors. Thefurther agent may also be any other non-direct acting antiviral agent,such as an interferon or other immunomodulatory agent.

Also disclosed are methods of modulating hepatitis B virus covalentlyclosed circular DNA comprising contacting a hepatitis B virus with anagent that provides epigenetic modification of the cccDNA, a histonemodifying agent, or an inhibitor of histone deacetylase activity. Forexample, the epigenetic modifying agent, histone modifying agent, orinhibitor of histone deacetylase activity may be pharmacological, suchas a small molecule. The epigenetic modifying agent, histone modifyingagent, or inhibitor of histone deacetylase activity may be selective forthe inhibition of cccDNA, as compared with integrated HBV DNA, i.e.,does not inhibit integrated HBV DNA, and/or as compared with cellularhost DNA, i.e., does not inhibit cellular host DNA. The inhibitor ofhistone deacetylase activity may be an inhibitor of multiple classes ofhistone deacetylase, or may be selective for a particular class ofhistone deacetylase. For example, the inhibitor may be an inhibitor ofclass I histone deacetylase activity, class II histone deacetylaseactivity, or both. Preferably, the inhibitor of histone deacetylaseactivity is an inhibitor of class I histone deacetylase activity.Numerous inhibitors of histone deacetylase activity are known, and anysuch HDAC inhibitor may be used pursuant to the present methods.

In accordance with the present methods of modulating hepatitis B viruscovalently closed circular DNA, the inhibitor of histone deacetylaseactivity may be, for example, Trichostatin A, suberoyl bis hydroxamicacid, 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl] benzamide,Apicidin, an Apicidin analog (for example, a natural analog of Apicidinor an analog that is synthesized de novo), or a compound according toformula (I)

wherein

R₁ is —(CH₂)_(n)— or —C(═O)—;

R₂ is —C(═O)—, 3,5-triazolyl, or —C(Z)N(R₄)—;

R₄ is hydrogen, alkyl, aryl, aralkyl, dialkylaminoalkyl, orcarboxyalkyl;

R₃ is —CH(R₅)—, or R₂ is nitrogen and R₃ is —CH— and R₂ and R₃ togetherform piperidinyl;

R₅ is hydrogen, —CH₃, or an alpha amino acid R group;

R₆ is —(CH₂)_(m)C(X)Y, —(CH₂)₂CH₃, or—(CH₂)_(q)-phenyl-(CH₂)_(m)C(═O)NHOH;

X is ═O, H₂, ═N—NH₂, or ═N—NH—C(═O)NH₂;

Y is NHOH or —CH₂CH₃;

Z is H₂ or O;

R₇ is hydrogen or alkoxy;

R₈ is alkyl or carboxyalkyl;

n is 0-2;

m is 0-6; and,

q is 0-3;

or a stereoisomer or pharmaceutically acceptable salt thereof.

In certain embodiments, the inhibitor of histone deacetylase activity isApicidin,

wherein

-   -   R₁ is —(CH₂)—,    -   and,    -   R₂ is —C(Z)N(R₄)—        or a stereoisomer or pharmaceutically acceptable salt thereof.

In other embodiments, the inhibitor of histone deacetylase activity is

or a stereoisomer or pharmaceutically acceptable salt thereof.

The present methods of modulating hepatitis B virus covalently closedcircular DNA may also include—in addition to the contacting of ahepatitis B virus with an agent that provides epigenetic modification ofthe cccDNA, a histone modifying agent, or an inhibitor of histonedeacetylase activity—contacting the hepatitis B virus with atherapeutically effective amount of a further agent that modulateshepatitis B virus. The contacting of the further agent with the HBV mayoccur simultaneously with, or simply as a part of the same procedurethat involves contacting the HBV with the agent that provides epigeneticmodification of the cccDNA, histone modifying agent, or inhibitor ofhistone deacetylase activity. The further agent may be any substancethat is presently used for modulation of HBV, of which numerous typesare known among those skill in the art. For example, existing drugs forthe modulation of HBV include interferons (e.g., interferon alpha,pegylated interferon), nucleoside analogues (e.g., lamivudine, adefovirdipivoxil, entecavir, telbivudine, tenofovir, clevudine, amdoxovir),non-nucleoside antivirals (e.g., BAM 205, ANA380, myrcludex B, HAPCompound Bay 41-4109, REP 9AC, nitazoxanide, dd-RNAi compound, ARC-520,NVR-1221), non-interferon immune enhancers (e.g., thymosin alpha-1,interleukin-7, DV-601, HBV core antigen vaccine, GS-9620, GI13000), andpost-exposure and/or post-liver transplant treatment drugs (e.g.,hyperHEP S/D, Nabi-GB, Hepa Gam B).

In particular, the further agent may be any other Direct ActingAntiviral anti hepatitis B agent (such as the polymerase inhibitorsBarraclude, Tenofovir, lamivudine, telbivudine, and adefovir) and/or anyother directing acting antiviral agents that work at a step in the viruslife cycle other than suppression of cccDNA transcription, such ascapsid inhibitors, secretion inhibitors, or entry inhibitors. Thefurther agent may also be any other non-direct acting antiviral agent,such as an interferon or other immunomodulatory agent.

The present disclosure also pertains to compound according to formulaII:

wherein

R₁ is —(CH₂)_(n)— or —C(═O)—;

R₂ is —C(═O)— or —C(Z)N(R₄)—;

R₄ is hydrogen, alkyl, aryl, aralkyl, dialkylaminoalkyl, orcarboxyalkyl;

R₃ is —CH(R₅)—;

R₅ is hydrogen, —CH₃, or an alpha amino acid R group;

R₆ is —(CH₂)_(m)C(X)Y, —(CH₂)₂CH₃, or—(CH₂)_(q)-phenyl-(CH₂)_(m)C(═O)NHOH;

X is ═O, H₂, ═N—NH₂, or ═N—NH—C(═O)NH₂;

Y is NHOH or —CH₂CH₃;

Z is H₂ or O;

R₇ is hydrogen or alkoxy;

R₈ is alkyl or carboxyalkyl;

n is 0-2;

m is 0-6; and,

q is 0-3;

or a stereoisomer or pharmaceutically acceptable salt thereof,

For example, the compound may be

wherein

R₁ is —(CH₂)—,

and,

R₂ is —C(Z)N(R₄)—.

In other embodiments, the compound may be

As will be readily understood, functional groups present may containprotecting groups during the course of synthesis. Protecting groups areknown per se as chemical functional groups that can be selectivelyappended to and removed from functionalities, such as hydroxyl groupsand carboxyl groups. These groups are present in a chemical compound torender such functionality in room temperature chemical reactionconditions to which the compound is exposed. Any of a variety ofprotecting groups may be employed with the present invention. Protectinggroups that may be employed in accordance with the present invention maybe described in Greene, T W. and Wuts, P. G. M., Protective Groups inOrganic Synthesis 2d. Ed., Wiley & Sons, 1991.

In a further aspect, the present disclosure relates to pharmaceuticalcompositions comprising a compound according to formula (I) or (II), ora pharmaceutically acceptable salt, isotopically substituted analogue,or stereoisomer thereof and a pharmaceutically acceptable carrier,diluent, or excipient. The applicable carrier, diluent, or excipient maybe selected on the basis of the chosen route of administration andstandard pharmaceutical practice as described, for example, inRemington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1985),the disclosure of which is hereby incorporated by reference in itsentirety. The pharmaceutical compositions may further comprise atherapeutically effective amount of a further agent that modulateshepatitis B virus. For example, the further agent that modulates virusmay be a known anti-viral agents. In certain embodiments, the presentcompositions comprise a therapeutically effective amount of a compoundaccording to formula (I) or (II) which is administered in combinationwith immunizations or vaccines that are effective in preventing orlessening the symptoms of HBV. Examples include antibodies, immunesuppressants, anti-inflammatory agents, and the like.

As used herein, the term “contacting” refers to the bringing togetherinto physical or chemical communication of indicated moieties in an invitro system or an in vivo system. For example, “contacting” an HBVvirus with a compound in the invention may include the administration ofa compound in the present invention to an individual or patient, such asa human, having an HBV infection, as well as, for example, introducing acompound of the invention into a sample containing a cellular orpurified preparation containing cccDNA.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, such as mice,rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses,or primates, such as humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician, which includes one or more of thefollowing:

(1) preventing the disease; for example, preventing a disease, conditionor disorder in an individual who may be predisposed to the disease,condition or disorder but does not yet experience or display thepathology or symptomatology of the disease;

(2) inhibiting the disease; for example, inhibiting a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,including arresting further development of the pathology and/orsymptomatology); and

(3) ameliorating the disease; for example, ameliorating a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., including reversing the pathology and/or symptomatology).

A subject or patient in whom administration of the therapeutic compoundis an effective therapeutic regimen for a disease or disorder ispreferably a human, but can be any animal, including a laboratory animalin the context of a clinical trial or screening or activity experiment.Thus, as can be readily appreciated by one of ordinary skill in the art,the methods, compounds and compositions of the present invention areparticularly suited to administration to any animal, particularly amammal, and including, but by no means limited to, humans, domesticanimals, such as feline or canine subjects, farm animals, such as butnot limited to bovine, equine, caprine, ovine, and porcine subjects,wild animals (whether in the wild or in a zoological garden), researchanimals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats,and the like, avian species, such as chickens, turkeys, songbirds, andthe like, i.e., for veterinary medical use.

The compounds of this invention may be administered orally orparenterally, neat or in combination with conventional pharmaceuticalcarriers, diluents, or excipients, which may be liquid or solid. Theapplicable solid carrier, diluent, or excipient may function as, amongother things, a binder, disintegrant, filler, lubricant, glidant,compression aid, processing aid, color, sweetener, preservative,suspensing/dispersing agent, tablet-disintegrating agent, encapsulatingmaterial, film former or coating, flavors, or printing ink. Of course,any material used in preparing any dosage unit form is preferablypharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations. Parenteraladministration in this respect includes administration by, inter alia,the following routes: intravenous, intramuscular, subcutaneous,intraocular, intrasynovial, transepithelial including transdermal,ophthalmic, sublingual and buccal; topically including ophthalmic,dermal, ocular, rectal and nasal inhalation via insufflation, aerosol,and rectal systemic.

In powders, the carrier, diluent, or excipient may be a finely dividedsolid that is in admixture with the finely divided active ingredient. Intablets, the active ingredient is mixed with a carrier, diluent orexcipient having the necessary compression properties in suitableproportions and compacted in the shape and size desired. For oraltherapeutic administration, the active compound may be incorporated withthe carrier, diluent, or excipient and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. The amount of active compound(s) in suchtherapeutically useful compositions is preferably such that a suitabledosage will be obtained. The therapeutic compositions preferably containup to about 99% of the active ingredient.

Liquid carriers, diluents, or excipients may be used in preparingsolutions, suspensions, emulsions, syrups, elixirs, and the like. Theactive ingredient of this invention can be dissolved or suspended in apharmaceutically acceptable liquid such as water, an organic solvent, amixture of both, or pharmaceutically acceptable oils or fat. The liquidcarrier, excipient, or diluent can contain other suitable pharmaceuticaladditives such as solubilizers, emulsifiers, buffers, preservatives,sweeteners, flavoring agents, suspending agents, thickening agents,colors, viscosity regulators, stabilizers, or osmo-regulators.

Suitable solid carriers, diluents, and excipients may include, forexample, calcium phosphate, silicon dioxide, magnesium stearate, talc,sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose,ethylcellulose, sodium carboxymethyl cellulose, microcrystallinecellulose, polyvinylpyrrolidine, low melting waxes, ion exchange resins,croscarmellose carbon, acacia, pregelatinized starch, crospovidone,HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminummethahydroxide, agar-agar, tragacanth, or mixtures thereof.

Suitable examples of liquid carriers, diluents and excipients for oraland parenteral administration include water (particularly containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil), or mixturesthereof.

For parenteral administration, the carrier, diluent, or excipient canalso be an oily ester such as ethyl oleate and isopropyl myristate. Alsocontemplated are sterile liquid carriers, diluents, or excipients, whichare used in sterile liquid form compositions for parenteraladministration. Solutions of the active compounds as free bases orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. A dispersioncan also be prepared in glycerol, liquid polyethylene glycols, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations may contain a preservative to prevent the growthof microorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form is preferably sterile and fluid toprovide easy syringability. It is preferably stable under the conditionsof manufacture and storage and is preferably preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier, diluent, or excipient may be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol and the like), suitablemixtures thereof, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of adispersion, and by the use of surfactants. The prevention of the actionof microorganisms may be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions may be achieved bythe use of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in the required amounts, in the appropriate solvent, withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions may be prepared byincorporating the sterilized active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation may include vacuum drying and the freeze dryingtechnique that yields a powder of the active ingredient or ingredients,plus any additional desired ingredient from the previouslysterile-filtered solution thereof.

The compounds of the invention may be administered in an effectiveamount by any of the conventional techniques well-established in themedical field. The compounds employed in the methods of the presentinvention including the compounds of formulas (I) or (II) may beadministered by any means that results in the contact of the activeagents with the agents' site or sites of action in the body of apatient. The compounds may be administered by any conventional meansavailable.

Preferably the pharmaceutical composition is in unit dosage form, e.g.as tablets, buccal tablets, troches, capsules, elixirs, powders,solutions, suspensions, emulsions, syrups, wafers, granules,suppositories, or the like. In such form, the composition is sub-dividedin unit dose containing appropriate quantities of the active ingredient;the unit dosage forms can be packaged compositions, for example packetedpowders, vials, ampoules, prefilled syringes or sachets containingliquids. The unit dosage form can be, for example, a capsule or tabletitself, or it can be the appropriate number of any such compositions inpackage form. In addition, dosage forms of the present invention can bein the form of capsules wherein one active ingredient is compressed intoa tablet or in the form of a plurality of microtablets, particles,granules or non-perils. These microtablets, particles, granules ornon-perils are then placed into a capsule or compressed into a capsule,possibly along with a granulation of the another active ingredient.

The dosage of the compounds of the present invention that will be mostsuitable for prophylaxis or treatment will vary with the form ofadministration, the particular compound chosen and the physiologicalcharacteristics of the particular patient under treatment. Generally,small dosages may be used initially and, if necessary, increased bysmall increments until the desired effect under the circumstances isreached. Generally speaking, oral administration may require higherdosages.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations. The dose may also be provided by controlled release ofthe compound, by techniques well known to those in the art.

Additional information regarding the preparation of the presentcompounds for administration and the formulation of compositionsaccording to the present invention is provided infra.

The compounds useful in the methods of the present invention may beprepared in a number of ways well known to those skilled in the art. Thecompounds can be synthesized, for example, by the methods as describedbelow, or variations thereon as appreciated by the skilled artisan. Thereagents used in the preparation of the compounds of this invention canbe either commercially obtained or can be prepared by standardprocedures described in the literature. All processes disclosed inassociation with the present invention are contemplated to be practicedon any scale, including milligram, gram, multigram, kilogram,multikilogram or commercial industrial scale.

For compounds herein in which a variable appears more than once, eachvariable can be a different moiety selected from the Markush groupdefining the variable. For example, where a structure is describedhaving two R groups that are simultaneously present on the samecompound, the two R groups can represent different moieties selectedfrom the Markush group defined for R.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment. Conversely,various features of the invention which are, for brevity, described inthe context of a single embodiment, can also be provided separately orin any suitable subcombination.

The present invention is further described in the following Examples. Itshould be understood that these examples, while indicating preferredembodiments of the invention, are given by way of illustration only, andshould not be construed as limiting the appended claims. From the abovediscussion and these examples, one skilled in the art can ascertain theessential characteristics of this invention, and without departing fromthe spirit and scope thereof, can make various changes and modificationsof the invention to adapt it to various usages and conditions.

EXAMPLES

Modulation of HBV cccDNA

DHBV cccDNA in LMH Derived Dstet5 Cells is Efficiently Produced andTranscriptionally Active.

Most HBV producing cells lines produce HBV gene products from an HBVtransgene integrated into the host chromosome, and thus cccDNA is notthe major source of viral product. This makes screening for drugs thattarget cccDNA difficult. Cell lines were produced in which viral geneproducts are dependent upon cccDNA. It was established human Hep G2 andchicken hepatoma (LMH)-stable cell lines for this purpose withtetracycline (tet) regulated HBV/DHBV. As shown in FIG. 1, after culturein the absence of tet and presence of 2 mM of foscarnet (PFA) to blockviral reverse transcription, DHBV RNAs accumulate, but DHBV replicationis arrested at the stage of pgRNA-containing nucleocapsids (lane 0).Upon addition of tet back to media to block transgene transcription, andremoval of PFA to allow the viral DNA synthesis in the pgRNA-containingcapsid to proceed, there is a rapid decline of viral RNA (day 1 and 2),with an eventual increase to a higher level when cccDNA is made afterday 3.

These results imply that cccDNA is efficiently formed andtranscriptionally functional in dstet5 cells. These results are morethoroughly demonstrated in FIG. 2, where, under the conditions specifiedin which the transgene transcription is blocked with tet, appearance ofnew HBV RNA is closely associated with appearance of cccDNA (FIG. 2Group II, core DNA shown), whereas, viral transcripts are rapidlydegraded (½ life˜3 hrs) in cells in which both cccDNA synthesis and newtransgene transcription is blocked (FIG. 2, Group I)

Identification of Compounds that Potently Repress DHBV cccDNATranscription.

With a system and conditions under which viral transcripts are producedin a cccDNA dependent manner (FIGS. 1, 2B), approximately 100 compoundswere screened, including those from the inventors' in-house smallcompound library, those present in the inventors' Natural Productscollection, and selected compounds including inhibitors of cellularepigenetic modification enzymes, including HDACs, HATs, Sirtuins,histone methyltransferases, histone demethylases and DNAmethyltransferases. Numerous compounds, including the four compoundsshown in Table 1, significantly reduced the amounts of cccDNA-derivedDHBV pgRNA. All possess HDAC class I inhibitory activity.

TABLE 1 Compounds that repress HBV cccDNA function and their activityagainst HDACs¹ 4-(dimethylamino)- N-[7- Suberoyl bis (hydroxyamino)-7-hydroxamic oxoheptyl] Hit Apicin Trichostatin A acid (SBHA) benzamide(M344) HBV cccDNA 0.183 0.480 2.50 6.25 (EC₅₀, uM)Toxicity >20.00 >20.00 >40.00 >100.00 (CC₅₀, uM)² SelectivityIndex >100 >40 >16 >16 (SI)³ HDAC-I YES^(4,5) YES^(5,6,9) YES^(4,6,7,10)YES^(4,7) inhibitor? HDAC-II NO^(3,7) YES^(4,5,6,9) YES & YES &inhibitor? HDAC III¹¹ HDAC III^(7,11) ¹Compounds found to suppress HBVcccDNA function in the dstet5 system, described in Prelim Evid., asillustrated in FIG. 2. ²Toxicity from our assays on dstet5 cells, as intext; Selectivity Index (SI) is the toxicity CC₅₀ divided by theEffectiveness EC50, see text. ³Selectivity Index (SI) is concentrationthat reduces 50% of cell viability (CC₅₀) divided by the concentrationthat reduces 50% of the HBV specific signal (RNA and/or HBeAg) (EC₅₀).⁴(7); ⁵(15)(26); ⁶(45); ⁷Reaction Biology Monograph; ⁸(33); ⁹(13);¹⁰(14); ¹¹(38)

Structures

As shown in FIG. 3, since Apicidin potently inhibited cccDNA (EC50˜180nM), with no toxicity at up to 20 uM for five days, and has nanomolaractivity against class I but not class II HDACs, it appears that HDAC IIinhibition is not necessary to suppress HBV in this system.

Apicidin and TSA Repress HBV cccDNA Transcription.

FIG. 3 shows that Apicidin and TSA repress cccDNA transcription inDstet5 cells. Evidence was also obtained demonstrating that thesecompounds also repress HBV cccDNA transcription in the HepG2 cells. Inmarked contrast, it was observed that Apicidin and TSA dose-dependentlystimulate DHBV pgRNA transcription from transgene integrated in hostcellular chromosome (FIG. 4). This is more typical of cellular generesponses to HDAC inhibitions and suggests that unlike chromosomal DNA,transcription from cccDNA “minichromosomes” are regulated differently.Moreover, there is even evidence that cccDNA levels were reduced,indicating, as seen in the Duck system, that transcriptional repressionis followed by destabilization.

Effect of Compounds Upon HBVcccDNA Transcription in Human HepatomaCells.

HepDE19 cells are seeded into 6-well plates, cultured in the presence oftetracycline until confluence. Tet is removed from the culture media toallow pgRNA transcription, DNA synthesis and cccDNA formation to occur.Tet is added back to culture media to shut off transgene transcription.After day 3, the cells, in different wells, are left untreated ortreated varying concentrations (i.e., 0.1 to 10.0 uM) of each of the“Test” compounds (four “hits” from Table 1 and ˜20 analogues) for 2days. Intracellular HBV cccDNA, viral RNA and core DNA are quantified bySouthern/Northern blot hybridization assays as described above and inknown procedures. Intracellular full-length HBeAg precursor and secretedHBeAg are quantified with Western blot and ELISA assays respectively.HepG2.2.15 cells are used as a control, because all HBV expression isprimarily from the HBV transgene in these cells. Interferon alpha, whichhas been shown to inhibit cccDNA transcription, and disubstituted-sulfonamides (DSS) CCC-0975, which inhibits cccDNA formation(from our screen, Guo 2012) will be included as positive drug controls.In some experiments, cultures are maintained for varying times (days)after removal of “Test” drug from the culture medium, to determine thedurability of any drug induced repression of HBV cccDNA. In order todetermine the selectivity of the testing compounds on cccDNAtranscription, effects of the testing compounds on the expression of apanel of cellular genes, including, but not limited to, alpha1antitrypsin, albumin, are also measured by quantitative RT-PCR orNorthern blot hybridization. The cytotoxicity of the compounds aredetermined by MTT assay in parallel cultures.

The amount (0-100%) of reduction of HBeAg and HBV transcripts is takenas a measure of HBV cccDNA transcriptional repression. The amount(0-100%) of HBV cccDNA reduction is taken as a measure ofdestabilization and degradation of HBV cccDNA. The amount (0-100%) ofrepression of A1AT and/or albumin mRNA reduction is taken as a measureof cellular function inhibition in specificity determination. The amountof MTT (0-100%) activity is taken as a measure of cell viability and thebasis of cell cytoxicity (CC). The Selectivity Index (SI) is as in theTable 1 legend.

Effect of Compounds Upon WHV cccDNA Transcription in Primary WoodchuckHepatocytes.

Woodchuck Hepatitis Virus (WHV) has been a useful model for evaluatingtherapeutics for HBV. Therefore, it is useful to know, for planning, ifthe lead compounds are active against WHV. Primary woodchuck hepatocytescultures (PWHCs) are prepared by plating collagenase treated tissues,derived from small section biopsies obtained from chronically infectedwoodchucks, under conditions where 50-90% of the hepatocytes harbor WHV,and the cultures (90% or greater) are hepatocytes and can be maintainedfor at least two months in culture, as done previously (see Fletcher SP, et al. 2012. Transcriptomic analysis of the woodchuck model ofchronic hepatitis B. Hepatology: In press). Within 7 days of seeding,cultures are incubated in the absence or presence of test compounds, andthe amount of WHV gene product in the culture medium (WHV virionassociated DNA; WHs) and intracellularly (WHV DNA, WHV RNA transcripts)are determined, using the similar methods as previously used andpublished (Guo, H., et al. 2010. Production and function of thecytoplasmic deproteinized relaxed circular DNA of hepadnaviruses. JVirol 84:387-396; Guo, et al. 2011. Alkylated porphyrins have broadantiviral activity against hepadnaviruses, flaviviruses, filoviruses,and arenaviruses. Antimicrob Agents Chemother 55:478-486)

Where WHV is sensitive (as suspected) to Apicidin and other candidatecccDNA inhibitors, at SI's similar to that in the avian and humansystems, then chronically infected woodchucks are used for an in vivoproof of efficacy study.

The inhibitors are ranked (i) by their selectivity index (SI), with themost selective in inhibiting HBV cccDNA transcription versus cellularviability, and then cellular function, being the most attractive; (ii)by their potency of inhibiting cccDNA transcription (lowest EC50) andfinally, (iii) by “critical chemistry” (scalability type/formulation)issues. The compounds with the lowest values of EC₅₀ (concentration thatinhibits 50% of the cccDNA-transcribed RNA) and greatest SIs, are themost attractive.

Identification of HDAC Isoform

Each of the compounds identified in the primary screen share theproperty of having HDAC inhibitory activity (see, e.g., Table 1). It islikely that HDAC inhibition is either part of, or central to, themechanism of the HBV antiviral action of these compounds. Although it isnot necessary to precisely know the compound's mechanism, thisinformation would be helpful in selecting or designing modifiedcompounds, as well as in forecasting and reducing possible in vivotoxicities, and designing clinical studies. Also, since crystalstructures are available for many HDACs, future drug design can beassisted. Taken together, the growing experience with HDAC inhibitors inresearch and in people, can could provide direction for the presentclinical designs and future plans.

HDACs deacetylate polypeptides (i.e., histones) and are classified intofour categories, based on function and DNA sequence homology. Class Iand II HDACs are inhibited by trichostatin A (TSA). Apicidin efficientlyinhibits class I, but not class II HDACs. Class III HDACs, calledsirtuins, are a family of NAD+-dependent proteins, not affected by TSA.Class IV is considered an atypical category, based on DNA sequence.Because Apicidin and TSA potently inhibited cccDNA transcription, HDACisozymes in classes I and II are most relevant. However, since Apicidininhibits only Class I, the initial focus is only on this class.

Experimental details: Silencing of HDAC isozyme transcripts with shRNAand the effect upon HBV cccDNA function. Short hairpin RNAs (shRNAs)expressed from lentivirus transduction vectors are now standard tools torepress translation of the transcripts to which the shRNA is homologous.A focus is placed on the class I HDAC isozymes. Therefore, confluentmonolayers of HepDE19 cells expressing HBV gene products in a cccDNAdependent manner (as described supra, after tet repression) aretransduced with 100 ul of lentivirus (˜5×10⁷/ml) in transductionmixture, expressing shRNA selective for class I-1, 2, 3 or 8 isozymesunder conditions where at least 95% of the cells receive and express theshRNA. This is determined by expression of reporter from the retroviraltransgene. The shRNA lentiviral expression vectors are provided by thevendor as transduction ready, and each vector targets different HDACs ofsub-class I. They are purchased from vendors (e.g., Santa Cruz Bio,OpenBio), and contain and express short hairpins with 19-25 ntshomologous to each HDAC isozyme transcript to be targeted. For example,one HDAC 1 specific shRNA contains 5′-GAT CCC CGC AGA (SEQ ID NO:1) . .. ATC TGC TTT TTG GAA A (SEQ ID NO:2)-3′, and others are similarlydesigned but specific for the other shRNAs, as provided by vendor andfrom previous work. There is 4- and 5-fold coverage for each HDACisozyme. Control vectors contain scrambled sequences, and are used asnegative controls. After 5 days of shRNA lentiviral transduction,repression of the specific HDAC is quantified by RNA analysis andwestern blot (with HDAC specific probes and monoclonal antibodiesprovided by vendor), and the amount of HBV cccDNA and cccDNA dependenttranscript, and HBeAg, is measured as was performed in precedingdescription. Where transient transduction approaches are unsatisfactory,although a bit more involved, stable transductions are used, since shRNAconstructs, with selectable markers are used. The amount of cellulargene expression (A1AT and albumin mRNA) are also quantified, asdescribed above, as a specificity control. Positive controls includeincubation of the HepDE19 cells with Apicidin at 1000 nM, aconcentration which represses HBV cccDNA (and will have been validatedon HepDE19). Each of the different HDACs has been associated withspecific cellular functions (i.e. 1, up regulation of p53, 2 & 3 p21, 8,deacetylation of H4) which are quantified as evidence of successful HDACsub class inhibition, should that be desired.

Given the potency of Apicidin, it is expected that silencing at leastone of the Class I HDACs will result in significant repression of HBVcccDNA function. It is recognized that the HDAC inhibitors, which act onmultiple HDACs, may have a greater effect than can be achieved by asingle HDAC transcript knock down. However, we knock down experimentsare also performed using lentivirus combinations covering all class Ienzymes, since this should repress HBV cccDNA if class I enzymes areinvolved (as Apicidin suggests), but multiple enzymes must be repressedto detect the HBV cccDNA inhibition.

Where silencing a specific or group of HDAC transcripts results inrepression of HBV cccDNA function, this is validated as a target for HBVantiviral action, and corroborated the finding that a mechanism ofanti-HBV cccDNA action of the identified compounds involves HDACinhibition. The compounds could, of course, use other mechanisms for HBVcccDNA suppression, but it will at least be known that HDAC inhibitiondoes repress HBV cccDNA, and the door is now open for this new class ofHBV therapeutic strategies.

Determining which of the Identified Compounds have the GreatestInhibitory Effect Upon the HDACs Isozymes Responsible for HBV cccDNARepression.

Having identified specific HDAC isozymes that are responsible forregulating HBV cccDNA, it is useful to identify the compounds that havethe greatest selectivity for inhibiting the HBV cccDNA regulatingisozyme. This allows for advancing the compounds with the greatestselectivity and help avoid off target effects resulting from needlesslyinhibiting HDAC isozymes that are not involved in regulating HBV cccDNA.We note that of the four compounds denoted above in Table 1, Apicidinhas the greatest selectivity index, and is also the one with thenarrowest HDAC inhibitory profile (selective for HDAC class I).Therefore, it is possible to achieve even greater selectivity byavoiding broad HDAC inhibitors and zooming in on the specific HDACsub-isozyme that is sufficient to repress HBV cccDNA.

Enzyme assays for each of the HDAC class I (1,2,3,8) isozymes areavailable as commercial kits, with positive and negative competitiveinhibitor controls. Kits are purchased corresponding to the relevantisozyme as identified above, from BioTeK, BPS Bioscience, or otheravailable sources. Briefly, with the BioTek system, sub class specificpurified HDAC enzyme (recombinant, at ˜10-50 ng/vessel) is provided,with a fluorogenic substrate, detected following deacetylation, withdeveloper in a premixed reaction. The enzymes that were shown bysilencing to be involved in HBV cccDNA repression are purchased. Varyingamounts of control or each of the experimental compounds are incubatedwith the enzyme reaction mix

The assay read-out is optimized for linearity both as a function of timeand enzyme concentration. Kits from the concentrations of the testingcompounds required to inhibit 50% of the deacetylase activity of an HDACisoform (i.e. IC50) are calculated by regression analysis usingSigmaPlot software (Systat Software, Inc., San Jose, Calif.).

Ideally, and most logically, compounds found to be active according tothe procedures described above are active against HDACs found to be mostinvolved in HBV cccDNA regulation, and these represent the favoredcompounds. Compounds that are active but broadly inhibit HDACs, some ofwhich are found to be irrelevant to HBV cccDNA regulation, are somewhatless favored, since they may bring unnecessary side effects. Where, onthe other hand, there is a disconnect, and the compounds active in thepreceding assays do not inhibit the HDACs found to be most important toHBV cccDNA regulation, the compounds are advanced based on HBV cccDNAsuppressive activity, and not HDAC inhibitory ranking.

Evaluation of Lead Compounds for their In Vitro Absorption,Distribution, Metabolism and Toxicity (ADMET) Properties HBV ProducingCells and Non Producing Cells

Introduction and Rationale

In vivo experiments are expensive and ethically constrained. Beforetesting in animals, it is therefore prudent to initially profilecompounds for potential toxicity and other cell-serum—interactiveproperties that are, to the extent possible, predictive of in vivoperformance. These studies have become standards in the field. Toxicityin replicating cells has also been found to be a good way to rankcompounds with respect to toxicity. Finally, differing formulations arealso usually necessary, before moving on to in vivo work, becausesolvents used in the tissue culture setting are not always compatiblewith in vivo administration. These are used, as below. An innovation inin vitro “ADMET” is presently proposed, in which the profiling iscarried out with HBV producing cells in the presence of a currentlyapproved antiviral therapies, in addition to the routine ADMET.

It is likely that new anti-HBV drugs, will be used in combination withthe other HBV antiviral drugs, in current use. Combination therapy isstandard for HIV and HCV and other infectious diseases. It is importantto know if a new drug to treat HBV has toxicities or other alteredprofiles in the presence of the current standards of care, since thereis evidence that many otherwise well tolerated medications haveselective toxicities in chronically infected individuals. HBV producingcells may be more sensitive to some emdications than are non producingcells (Block, in progress). Therefore, the toxicity experiments, below,are carried out in the absence as well as the presence of HBV polymeraseinhibitors and, in some cases, interferon alpha (IFNa).

Some of the present lead compounds may have already been used in animals(by others), there may be considerable information available. On theother hand, some of the leads may be new compounds for which there is noanimal data. Compound profiles are also examined in the context of HBVinfection, for the reason stated above.

Finally, compounds that suppress wild type HBV cccDNA function and arewell tolerated in vitro are tested for their ability to suppress cccDNAfrom HBV that is resistant to HBV polymerase inhibitors. Depending onthe results of the preceding studies, human and/or duck HBV transfection(and for the duck, infection) systems are used.

For every experiment described below, controls with known toxicity,metabolism, protein permeability, membrane transport and definedformulation properties are included. For example, Barraclude and FIAUare included as controls for compounds that have no detectable toxicityin HBV producing cells, and those that do, respectively, and havereported PK and TK properties for which comparisons can be made.

Experimental Detail: In Vitro “Administration, Distribution, Metabolism,“Elimination” and Toxicity” (ADMET) Studies.

Some of these experiments are carried out under contract by a Vendor(i.e. Absorption Systems) and others, particularly where HBV producingcells and material are used, are carried out by the present inventors,as indicated, below.

Standard Cytoxicity Assays.

Human hepatoma (HepG2, Huh7, HepRG) and HepG2-derived cell linessupporting constitutive (HepG2.2.15) and tetracycline-inducible HBVreplication (HepDE19 and HepDES19) are seeded into 96-well plates at adensity of 2×10⁴ cells per well. Cells are treated with a serialdilution of testing compounds. The culture media is changed every otherday. MTT assays are performed at day 2, 4, 6, 8 and 10 day sincetreatment.

Toxicity to Multiplying Cells:

Varying concentrations of lead compound(s) are incubated with HepRGcells seeded at low density (100 cells per well of 32 mm dish) under HBVproducing and non producing conditions, and cultured for 10 days, withmedia changes every 3 days.

Metabolic Stability in Human and Mouse Liver Microsomes:

The compounds are incubated with human and mouse liver microsomes fromHBV producing and non producing cells (tissue culture source as above)in the presence of NADPH. In addition, the stability of compounds areevaluated in the presence of human simulated gastric fluid and simulatedintestinal fluid. The purpose of this set of experiments is also todetermine if the compounds are metabolized by the digestive enzymes.Since orally available compounds are pursued, it is important to findout what metabolites, if any, might be produced in the GI tract.

The toxicity and metabolic stability studies are carried out in theabsence and presence of concentrations of lamivudine, barraclude,telbivudine, tenofovir and/or adefovir that are equal to and multiples(˜0.1 ug/ml, for barraclude, −10 ug/ml for lamivudine) or interferonalpha (IFNa) of the serum levels typically achieved in people. ThecccDNA suppressive test compounds are used at 10 times their IC50, asdetermined in assays described above. Control compounds (withestablished toxicities and established metabolic profiles) are alsoincluded with each panel of tests (i.e. FIAU, statins, etc).

Plasma Protein Binding:

Equilibrium dialysis is used in this assay to determine the percentageof compound that binds to human plasma proteins (by Vendor).

Bidirectional Permeability:

This assay is used to determine the permeability of compounds throughCaco-2 cell monolayers in the apical-to-basolateral andbasolateral-to-apical direction. (Contractor)

Antiviral Activity of Lead Compounds in the Presence of Interferons(IFNs).

The experiments above explore the in vitro ADMET of the lead compoundswhen used in combination with polymerase inhibitors or interferons inuninfected cells. It is also important to determine if the leadcompounds have an impact upon an established antiviral agent's antiviralproperties. Compared with pol inhibitors, IFN alpha (a) is lessfrequently used to manage HBV. When used, it is only for a period ofmonths, unlike pol inhibitors, which are used for years and more likelyto be co-administered with a cccDNA inhibitor. However, given the factthat IFNa mechanisms of antiviral action and toxicities may involveHDACs, it does make sense to evaluate the presently disclosed cccDNAinhibitors for their interaction profiles with IFNa, to the extent thiscan be evaluated in vitro. Therefore, the dSTET cells and AD38 cellsprogrammed to produce transcripts from HBV cccDNA (as in prelim evidenceand Cai 2012) seeded at cloning densities (for growth studies) and semiconfluence (for antiviral/cccDNA transcription studies) are incubated inthe absence and presence of varying concentrations of candidate cccDNAinhibitor and the absence and presence of amounts of either avian IFN orhuman IFNa known to suppress HBV in vitro. Cell viability and the amountof HBV cccDNA derived gene products (transcripts) produced aredetermined as in previously described procedures and those known in theliterature.

The compounds are also tested for in vitro activity in the presence ofthe currently used polymerase inhibitors. The emergence of mutantviruses resistant to the nucleoside/tide inhibitors of the HBVpolymerase is a problem in the management of chronic infection, althoughthe problem varies with the polymerase inhibitor used. Thus, compoundsthat suppress wild type HBV cccDNA function are tested for their abilityto suppress cccDNA from HBV that is resistant to HBV polymeraseinhibitors. All of the mutant viruses (DHBV and WHV) needed areavailable. Human and/or Duck HBV transfection (and for the Duck,infection) systems are used. Given the distinct mechanism of action, thepresent compounds retain antiviral activity.

Formulation Optimization:

For selected compounds, dosing vehicle development suitable for oralgavage are evaluated. The test vehicles include 1) pH manipulation, 2)co-solvents (such as glycin, polyethylene glycol propylene glycol,ethanol etc), 3) surfactants (such as polysorbates, polozamer, polyoxylcastor oil, glyceryl and PEG esters), 4) Non-aqueous systems (such assesame oil, medium chain triglycerides, soybean oil, oleic acid), 5)complexing agents (such as cyclodextrins).

From an ADMET perspective, preferred are compounds that have propertiessimilar to Barraclude, with respect to tolerability. Also preferred arecompounds that have the same toxicity and metabolic stability profilesin the absence of HBV polymerase inhibitors (lamivudine, barraclude,interferon etc) as in their presence. Compounds with selective toxicityto HBV producing cells are disfavored, disqualified, or advanced withextra caution. Compounds that have enhanced, or enhance, the toxicity ofcurrent HBV antivirals, or antagonize the antiviral, activity thosecompounds, are still advanced, but with caution and tested in in vivoexperiments for the possibility of enhanced toxicity in combination. Itis possible to propose that the cccDNA active compounds not be used (oronly used cautiously) in combination.

Lead Compounds with Favorable In Vitro Properties are Scaled Up andTested for In Vivo Toxicity, Pharmaco Kinetics (PK) and Efficacy

Pharmacokinetic, Toxico-Kinetic (TK), and Dose Range Finding Studies.

Prior to conducting in vivo efficacy studies, which are expensive,ethically constrained, and consume great amounts of compound, it isnecessary to determine the maximum tolerated doses (MTDs) andpharmacokinetic properties (PK) of the candidate drugs, in vivo, inuninfected animals. This permits the identification of compounds worthyof advancement and establish proper dosing and routes of administration.Compounds are tested for efficacy in either (or both) duck and/orwoodchuck models of chronic hepadnavirus infection, since these are theestablished and predictive animal models. The rationale for duck versuswoodchuck is described below. Regarding Apicidin itself, a great dealwill already be known about its PK/TK in animals, since it has alreadybeen used in mice. However, even for Apicidin, and certainly for anyother of the present compounds, new PK, TK for the Duck and woodchuckstudy are needed. Therefore, a series of murine and rat PK and TKstudies are conducted as follows.

Experimental Detail—Single Dose Pharmacokinetic Study in Mice, Ducksand, if Indicated, Woodchucks.

The objective of this study is to obtain volume of distribution,systemic clearance, half-life (T½), maximal plasma concentration (Cmax)and bioavailability. These parameters are used to evaluate the clearanceand bioavailability of each imino sugars so that the compounds can beranked by their ability to maintain plasma concentration. In general,greater than 50% bioavailability is preferred for compounds to beadvanced.

As described above, candidates are administered via i.v. injection (5mg/kg) or given orally (25 mg/kg) to mice (6 week old Balb/c; 6mice/group); Peking Ducks (6 week old) or woodchucks (3 per group).Clinical observations are recorded at several intervals after dosing.Blood and urine samples for pharmacokinetics are collected predose, andat 5, 15, and 30 min, 1, 2, 4, 6, 8, 16 and 24 h post-dose. Samples areanalyzed for the presence and amounts of administered drug (drug orprodrug) and in the case of administered prodrug, for the presence andamount of “drug” metabolite” as well. The samples are analyzed byAbsorption Systems, who has established mouse plasma assays for ourother compounds.

Tissue distribution (murine).

Tissue is taken from mice (3 per dose group) receiving a single oral oriv administration of compound at various times after administration.Knowledge of the tissue distribution of a compound can significantly aidin evaluating potential as successful drug candidate. Although other invitro parameters, such as plasma protein binding and volume ofdistribution have prediction values for rate and extent of distributionto extravascular tissues, the liver tissue concentration of drug isprobably most relevant to efficacy. A focus is maintained on liver, incomparison to serum, kidney and abdominal fat tissue/lymph nodes, fortissue concentration of candidates, using endpoint samples, followingthe single administration of the compound by an i.p. and oral route inmice. One point of interest is if active compound builds up in keytissue, which provides insights regarding its effective half life, intissue. That is, although the serum half life of a drug might be ˜2hours, it could have a tissue half life in liver several fold timesthat, explaining a greater than expected efficacy (for a given dosingregimen), or greater than expected toxicity.

Dose-Finding Maximum Tolerated Dose (MTD) Study.

Since the compounds are evaluated for antiviral activity in murinemodels, it is important to know the tolerability of the compounds inmice. Balb/c mice (6 week old, 6 per group); Ducks (6 week old, 3 pergroup) will be dosed by oral gavage (since we are pursuing orallyavailable compounds) either “vehicle” alone, or vehicle in whichcompound has been dissolved. From previous experience, the range ofcompound administered is likely between 100 mg/kg to 500 mg/kg, 5 miceper dose group. Animals will be observed for up to 14 days, with dailyreadings of weight and an endpoint of survivability. Routine histologyand clinical chemistry studies are be performed. The highest dose ofcompound that does not result in any mortality/toxicity is considered tobe the MTD. Woodchucks can not be used for this MTD study;extrapolations from the murine study, combined with the PK woodchuckstudy will be necessary.

The compounds are ranked for their oral bioavialibility, tolerability,and half lives. The ideal compound is able to reach and sustain serum orliver tissue levels at least 10 times the tissue culture IC50concentration, with soluble, oral, single day dosing, and have MTDs morethan 100 times that of the tissue culture IC50. Compounds are rankedwith respect to these qualities, and the best and second best will beadvanced.

Is the Lead Compound Efficacious in Chronically Infected Animal Models,In Vivo?

Having demonstrated in vitro efficacy, and determined safe and rationaledosing for in vivo work, it is be important to know if the leadcompounds can control viral levels in validated animal models of chronicHBV. This represents the first time a small molecule drug that targetscccDNA will have been tested in animals. Outcomes consistent with asafe, selective and cccDNA targeting agent are of interest. Efficacy endpoints include: rapid and coordinated reduction in viremia, antigenemiaas well as amount of intra-liver cccDNA and replicative forms whichwould be indicative of cccDNA suppression. These goals dictate theanimal models that are used, and length of treatment that is studied.

Several animal models of chronic HBV infection exist, and each hasvirtues as well as disadvantages. Ducks and woodchucks can beexperimentally chronically infected with duck and woodchuckhepadnavirus, respectively. There are now several murine models, butsince transgenic mice bearing HBV transgenes do not produce HBV fromcccDNA templates, to test a cccDNA targeting compound, a chrimeric mousewith human hepatocytes would be necessary, such as the uPA mice.Practical considerations require making a choice. Experiments aredesigned for evaluation in the Duck model of chronic HBV, since thecompounds are active against the Duck virus in avian cells in cultureare already known. Studies in the chronically infected woodchuck arealso prepared, since this is an established model for testing HBVtherapeutics and is a natural infection. The uPA mice are very expensivebut will are if woodchucks are not sensitive to the drugs, but human HBVis.

Therefore, preferred compounds are scaled up to the amount necessary andtested for efficacy, as defined below, in the following Duck, and ifappropriate, woodchucks.

Experimental Detail—Scale Up Production of Preferred Compounds.

Apicidins are produced in fermentations by Fusarium (i.e. sp. ATCC74322). The strain is inoculated into a nutrient medium called MED5,shaken at 220 rpm, for 12-16 days in a controlled humidity atmosphere.At harvest, whole broth is extracted with methylethylketone and theextract is fractionated by gel filtration on Sephadex followed by finalpurification by RP-HPLC. Yields are on the order of 250 mg/L so scale upto gram amounts are routine.

Duck Hepadnavirus Efficacy Study.

Since it is known that Apicidin is highly active against the DHBV, inculture, it is tested in a chronically infected duck. The goal of thisstudy is to determine the antiviral potential of preferred compounds.Serology and histology are secondary.

Six-week-old Peking Ducks, chronically infected with DHBV type 16(Alberta Strain), are used. At 6 weeks, viremia and liver mass in duckstends to have stabilized. Ducks are given, by either i.m. or oral gavage(depending on Aim 4 PK/TK results), test compound (3 dose groups, withdosing amount and frequency to depend on PK results, but aiming toachieve stable serum levels of at least 10 times the tissue cultureIC50). There are three dose groups with 5-6 animals per dose group.Control dose groups (6 animals each group) include placebo treatedanimals and animals treated with either barraclude (1 mg/kg) orlamivudine (40 mg/kg) per day. At least three animals from all dosegroups contribute at least one pre treatment and one post treatmentliver biopsy. Treatment is for 10 weeks, since this exceeds the time forlamivudine to suppress viremia to beneath detectable levels and thereported ½ life of cccDNA in the duck. Ducks are followed with weeklyserum collections for an additional 4 weeks after withdrawal of drug.Serum will be collected weekly.

Weekly serum is tested for standard “lab values” (hematology, albumin,AST, ALTs, The amount of DHBV viral DNA, sAg, sAb in the circulation isdetermined. Liver tissue derived from biopsies (some pre treatment andend of treatment from the same animals) is examined for DHBV DNA(cccDNA, replicative forms) and DHBV core (immunostained).

WHV-Infected Woodchuck Study.

The study uses 10 groups, with 5 animals per group, with drug treatmentfor 10 weeks followed by 10 weeks off drug (to test durability ofaffect). Due to variability in the levels of viremia and antigenemia,animals are stratified to groups by WHV viremia and antigenemia levelsas determined seven days prior to study start, so that the averagelevels of both viral markers are evenly distributed among all groups ofanimals. Animals with abnormally low WHVsAg levels are not used in thisstudy. Compound is administered daily, by a route and frequency to befinalized after bioavailability studies in rodents. The first day ofdosing on the study is Study Day 1. Study Day 1 dose levels arecalculated on a pretest body weight, and body weights are taken weeklyfor dose administration. Dosing range is as for the mouse study overfour doses, with Group 10 treated with Barraclude as a referencecompound (Tennant).

The primary endpoint is a dose dependent reduction in viremia andantigenemia on and off drug achieving durable off drug reductions.

Viability and Animal Health.

Clinical observations are performed and recorded once daily formorbidity and mortality. Further toxicology is addressed via hematology,serum chemistry, and histology examination. It is also important toconsider all biochemical and immunological endpoints in the context ofgeneral animal health to insure that decreases in viremia or antigenemiaor other putative beneficial outcomes are not a secondary consequenceprotocol (compound) toxicity. Gross physical characteristics (weight,stool and urine output and characterization, are determined on a weeklybasis. In addition, liver function tests (performed on samples collectedmonthly), hematology and chemistry (performed on pre, mid and end oftreatment samples (as described in the table) and, for selected animals(at pre-dose, mid dose and end of treatment times), histology on punchbiopsy derived liver sections are also performed for assessment oftoxicity as well as efficacy

Liver function test are determined by commercial service in the monthlysamples as a marker of liver viability

Evidence of Humoral Responsiveness.

The presence of antibodies that recognize WHsAg are determined by anELISA. This assay is such that even WHs Abs complexed with antigen aredetected.

Toxicology.

Careful toxicology is carried out via hematology and serum chemistry asdescribed for the mouse studies. In addition, histological examinationof the punch biopsies of the livers is undertaken, includinginflammation, bile duct proliferation, and portal and lobular hepatitis.

WHV Virus Levels in the Serum.

An assessment is performed on weekly (as slot blot hybridization and PCRor bi-monthly (southern blot).

Biopsies.

Liver biopsies are collected before the start, middle, end of treatment,and end of study and used for histology and intracellular WHV DNAexamination. Levels of replicative form and intrahepaticcovalently-closed circular WHV DNA (WHV cccDNA) are quantitativelydetermined based on Hirt extraction. For immunostaining, separate tissueis used and accumulation of core and WHsAg in treated versus untreatedanimals will be determined.

For both the Duck and woodchuck studies, no technical difficulties areexpected, since these studies are fairly routine, with all methods andreagents needed for evaluation being in hand. One possible problem withDucks is the variations in viremia/antigenemia that occur without drug.This is mitigated by using Ducks after 6 weeks of age, in which virologyas usually stabilized.

The benchmark of positive activity is LFMAU treated animals. Theseanimals are expected to have uniformly lost HBV viremia and evenantigenemia, by 3 and 10 weeks of treatment, in the Duck and Woodchuck,respectively, with numbers of HBV infected hepatocytes greatly reduced,relative to pretreatment and untreated groups.

Inhibition of cccDNA transcription (and stability) should reduce theintracellular and extracellular amounts of all viral gene products (at arate influenced by their serum half lives), even before there arereductions in the numbers of HBV infected cells (and possibly, out ofproportion to the number of HBV infected cell loss). Realistically, theclearest evidence of efficacy of our new compounds is time and dosedependent statically significant reductions in HBV DNA viremia and sAgantigenemia. Given the efficacy of the present compounds, in vitro, anat least a ten-fold reduction of serum surface antigen in either or bothmodels is expected.

DHBsAgWHsAb levels are also measured. Control, chronically infectedanimals are expected to have no detectable (or very little detectable)Ag. There is a growing body of evidence that chronically infected people(and woodchucks) are capable, and do make, sAb, but it is suppressed orbound with circulating sAg. It is therefore possible that if and as Agdeclines, sAb will declare itself.

Biopsy analysis is performed on immunostained for HBV core, sAg, usingmounted liver tissue, and with extracts to examine the amounts of HBVnucleic acid, before and after treatment. Ideally, the numbers ofinfected cells will decline as a function of drug treatment. Usefulinformation includes whether this occurs in a setting of increasedhepatitis (cell infiltration).

Serum from animals for 10 & 4 weeks (Woodchuck and Duck, respectively)is also evaluated after drug treatment has been stopped. Stable, offdrug, repression of antigenemia, viremia, with appearance of sAbs isconsidered the obtaining all major objectives. On drug suppression ofviremia and antigenemia by amounts exceeding placebo, in the absence ofany adverse reactions or events, is considered proof of a drug specificaffect.

The animal studies outlined above permit definitive conclusions as towhether the compounds are effective at reducing antigenemia in an invivo context.

Where inhibition of an HDAC is determined to repress HBV cccDNAtranscription, the results are as surprising as they are useful, sinceHDAC inhibition has generally been associated with gene activation,including HBV DNA integrated into host chromosomes. The results mayrepresent an example of how different is the regulation of HBV cccDNAfrom most cellular genes and, even if the inhibitors identified hereinare not ultimately used in human systems, it is demonstrated that it ispossible to non-catalytically inhibit cccDNA with small,pharmacologically, active compounds.

Taken together, this work delivers two very critical answers. First, itindicates the selective suppression of HBV cccDNA function in human andwoodchuck cultures. Second, it determines which HDAC (the target ofApicidin) regulate HBV cccDNA. We understand that HDAC inhibition in HBVinfected people must proceed with caution, and this work representsdirection regarding how to go proceed with a revolutionary newtherapeutic strategy.

General Synthesis

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or suitable process conditions(i.e., reaction temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are given, other process conditions can also be usedunless otherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C NMR), infrared spectroscopy (IR),spectrophotometry (e.g., UV-visible), or mass spectrometry, or bychromatography such as high performance liquid chromatography (HPLC) orthin layer chromatography.

Preparation of compounds can involve the protection and deprotection ofvarious chemical groups. The need for protection and deprotection, andthe selection of appropriate protecting groups can be readily determinedby one skilled in the art. The chemistry of protecting groups can befound, for example, in P. G. M. Wuts and T. Greene, Greene's ProtectiveGroups in Organic Synthesis, 4th. Ed., Wiley & Sons, 2006, which isincorporated herein by reference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected. The compounds of the inventioncan be prepared, for example, using the reaction pathways and techniquesas described below.

Compound Synthesis

Apicidins have been derivitized and recent analogs 1 and 2

(see Horne, W. S., C. A. Olsen, J. M. Beierle, A. Montero, and M. R.Ghadiri. 2009. Probing the bioactive conformation of an archetypalnatural product HDAC inhibitor with conformationally homogeneoustriazole-modified cyclic tetrapeptides. Angew Chem Int Ed Engl48:4718-4724; Vickers, C. J., C. A. Olsen, L. J. Leman, and M. R.Ghadiri. 2012. Discovery of HDAC Inhibitors That Lack an Active SiteZn2+-Binding Functional Group. ACS Medicinal Chemistry Letters)demonstrate that the Apicidin structure can be modified without loss ofanti HDAC potency.

Further analogs were prepared with a focus on improving pharmaceuticalproperties relative to Apicidin, which has very poor aqueous solubility,oral bioavailability, and half life in vivo. Apicidin derivatives wereprepared, inter alia, by standard solid and solution phase methods. Incertain embodiments, the reduced beta-isoleucine amino acid derivedfragments 4

were prepared in a suitably protected form (PG=suitable protectinggroup, such as Fmoc or Boc) by solution phase methods and introducedinto the amino acid sequence by solution or solid phase means, followedby cyclization using established methods.

What is claimed:
 1. A compound of formula II:

wherein R₁ is (CH₂)_(n)— or —C(═O)—; R₂ is —C(═O)— or —C(Z)N(R₄)—; R₄ ishydrogen, alkyl, aryl, aralkyl, dialkylaminoalkyl, or carboxyalkyl; R₃is —CH(R₅)—; R₅ is hydrogen, —CH₃, or an alpha amino acid R group; R₆ is—(CH₂)_(m)C(X)Y, —(CH₂)₂CH₃, or —(CH₂)_(q)-phenyl-(CH₂)_(m)C(═O)NHOH; Xis ═O, H₂, ═N—NH₂, or ═N—NH—C(═O)NH₂; Y is NHOH or —CH₂CH₃; Z is H₂ orO; R₇ is hydrogen or alkoxy; R₈ is alkyl or carboxyalkyl; n is 0-2; m is0-6; and, q is 0-3; or a stereoisomer or pharmaceutically acceptablesalt thereof.
 2. The compound of claim 1, which is

wherein R₁ is —(CH₂)—, and R₂ is —C(Z)N(R₄)—.
 3. The compound of claim1, which is


4. A pharmaceutical composition comprising at least one compound ofclaim 1 and at least one pharmaceutically acceptable carrier, diluent,or excipient.
 5. The pharmaceutical composition of claim 4, furthercomprising an additional anti-hepatitis B infection agent.